Cloned human skeletal muscle satellite cells (HMSC) will be used to analyze extracellular signals that control their growth and differentiation. Because of major differences among species, it is necessary to use human cells to gain a fully accurate understanding of such controls. A recently-developed serum-free growth medium for HMSC, consisting of optimize nutrient medium MCDB 120 plus epidermal growth factor (EGF), dexamethasone, insulin, fetuin, and serum albumin, will be used as the starting point for these studies. At present, growth is further enhanced by adding serum to the serum-free medium, and the exact roles of insulin, fetuin, and serum albumin remain uncertain because of the large amounts needed, the potential presence of impurities, and possible cross reaction of insulin with IGF-I receptors. The proposed research will clarify the exact requirements of HMSC for "mitogens" (in the broadest sense of the word), and as, each "mitogen" is precisely identified, its effects on both growth and differentiation will be analyzed in detail and compared to literature data for other species. It is already evident that human muscle cells have different mitogen requirements than myogenic animals cells. In our serum-free medium, which contains no fibroblast growth factor (FGF), HMSC exhibit a well-defined requirement for EGF. However, FGF is known to play a key role that cannot be replaced by EGF in growth of myogenic mouse cell lines and myoblasts from several other species, while the rat L6 line does not respond to either FGF or EGF, and required IGF-I. The effects of combinations of mitogens and of the composition of the nutrient medium on myogenic differentiation will also be examined, as well as the possibility that differentiation may be enhanced (or inhibited) by substances that do not alter growth. These studies are expected to dissociate initiation of differentiation at least partially from cessation of proliferation, and to provide new insight into complex relationships among growth factors, proliferation, and terminal differentiation of normal human muscle cells. As time permits, studies will also be initiated on second message systems that are affected by the extracellular signals that play primary roles in activation of differentiation of HMSC. The goal will be to trace the myogenic signals inward step-by-step until they meet up with studies in other laboratories starting form muscle-specific gene regulatory sequences and their associated trans-acting protein factors toward the primary control signals. This research will generate a new level of understanding of the mechanism that regulate human skeletal muscle growth and differentiation, which in turn will provide a better basis for analysis and treatment of the human muscle diseases, as well as rehabilitation of injured muscles.